PROJECT SUMMARY Motivated behaviors, included those altered in substance use disorders, are known to be regulated by monoaminergic circuits (containing neurons that release dopamine, serotonin, and norepinephrine) in multiple brain regions. Disentangling these circuits from the cell-level to the systems- and behavioral-levels has almost exclusively been limited to studying the physiology of neurons. However, recent data suggest that the activity of a non-neuronal cell type?astrocytes?may have profound effects on how monoaminergic networks function, but how astrocytes respond to monoamines and change their physiology in response to repetitive drug exposure remain almost completely unstudied. The current proposal aims to address gaps in our understanding of addiction, monoamine signaling, and multi-cell type population dynamics by uncovering fundamental cell biological signaling systems in astrocytes that are critical for the function of brain regions with monoaminergic input. In Aim 1, we will use two-photon imaging and photoactivation to interrogate the cell signaling mechanisms that prefrontal cortical astrocytes deploy to sense extracellular monoamine dynamics across cortical layers. In Aim 2, we will decode the mechanisms by which astrocytes change, both instrinsically and in their output to the neuronal population, after long-term exposure to a psychostimulant. With these aims, we will probe the roles of astrocyte physiology in baseline prefrontal cortex function and as mediators of circuit neuroplasticity in a model of addiction.